Overview of gene expression and gene regulation in eukaryotes Flashcards
what is the lac operon
a regulated prokaryotic promoter
- activated by CAP, repressed by lac repressor
- ideal conditions for expression = +lactose and -glucose
euchromatin vs heterochromatin
euchromatin: opened chromatin, active genes found here
heterochromatin: closed chromatin, inactive genes found here, not accessible to transcriptional machinery
what type of RNA does each RNA polymerase produce
RNA pol I: pre-rRNA
RNA pol II: mRNA, miRNA, snRNA
RNA pol III: tRNA, snRNA, 5S RNA U6, 7S RNA
what is epigenetic regulation
chromatin-mediated regulation - to transcribe DNA chromatin must be opened
what is the structure of eukaryotic polymerase II?
complexes of multiple polypeptides
- 12 polypeptides RBP1, RBP2 … RBP12
- RBP1 is the clamp domain which accommodates DNA and then is closed by a bridge
- contains a carboxy-terminal domain of CTD1 subunit
-
What is the carboxyl-terminal domain (CTD)
- involved in regulatory interactions in initiation, release, elongation and processing of mRNA
- phosphorylation of the CTD allows RNA pol II to move along the DNA
- Ser residue in the CTD are phosphorylated upon transition from initiation to elongation
- not structures - long and wobbly
What regulates transcribed genes?
- conserved basal promoter elements (core promoter sequences)
- promoter-proximal binding sites for activators
- distal enhancers and repressors
- chromatin structure
What are the core promoter sequences in DNA?
- TATA box - prevalent in highly transcribed genes
- Initiators - poorly conserved
- BRE or DPE - influence activity of promoter
What components help RNA pol recognize promoters and correctly initiate transcription
- several GTFs assemble the PIC over core promoter sequences
- DNA helicase helps initiate transcription
- Protein kinase helps release the polymerase
- other factors help the polymerase elongate
- other factors move nucleosomes
what are the general transcription factors of RNA pol II
TFIIA, TFIIB, TFIID, TFIIE, TFIIH
What are the similarities between TATA and CpG island promoters
- transcription proceeds in one direction
- RNA pol II is loaded onto them by GTFs
What are differences between TATA and CpG island promoters
TATA
- transcription initiates in only 1 direction
- more nucleosomes = harder to transcribe
CpG island
- transcription is initiated in both directions
- transcription initiates anywhere on the island
- less nucleosomes = easier to transcribe
what is ChIP
chromatin immunoprecipitation
- multistep technique that determines which DNA the DNA binding proteins is attaching to
- with anti-RNA pol II antibodies shows the binding of RNA pol II to the DNA
- shows 2 peaks for CpG islands
- shows 1 peak for TATA
How is the pre-initiation complex assembled?
- TFIID associates with the promoter followed by TFIIA and TFIIB
- the mediator facilitates joining RNA pol II to the GTFs
- RNA pol II attached to TFIIF is recruited, the non-phosphorylated CTD of RNA pol II establishes contact with several GTFs - now have the core PIC
- TFIIE and TFIIH are recruited - now have closed PIC
- assembly is now finished
How does the PIC open and become the initially transcribing complex?
- TFIIH helicase activity opens the DNA double helix
- RNA pol II initiates transcription
- TFIIH kinase activity phosphorylates the CTD to release RNA pol II from the promoter
- elongation can now commence
transition from transcription initiation to elongation
- after TFIIH phosphorylates CTD, 2 elongation factors NELF and DSIF associate with RNA pol II and pause it downstream of the initiation site
- at this point another kinase PTEFb (aka CDH9/CycT) phosphorylates the CTD and NELF to get rid of it
- once RNA pol is released elongation can begin
What does massive parallel RNA sequencing show
- the intensity of transcription over analyzed DNA
- tells you if we have synthesis of RNA in both sense and anti-sense strand
what are the 3 capabilities of TFIIH
- helicase activity
- kinase activity
- DNA repair - fix nucleotide mismatch
how can defects in transcriptional elongation lead to HIV
- HIV supresses PTEFb activity by a virally encoded protein called TAT
- TAT releases PTEFb and the polymerase transcribes the virus
How do NELF and DSIF pause only viral transcription? IMPORTANT
-NELF and DSIF pause the polymerase after initiation
- further phosphorylation of CTD by PTEFb releases the polymerase and elongation begins
- upon initiation at 5’ end viral DNA has TAR which host RNA doesn’t have
- TAR allows for the virally encoded protein TAT to associate with it and block the activity of PTEFb
- PTEFb can’t phosphorylate NELF or CTD so viral DNA transcription is held
- cell stress occurs and is sensed by TAT - TAT falls off
- normal transcription continues
What are the protein-binding transcription control regions that regulate gene expression
promoters: direct binding of RNA pol to DNA, multiple elements close to initiation site
promoter-proximal elements: close to promoter, inhibit transcription of genes
enhancers: increase transcription, could be upstream or downstream of initiation site
activators: boost gene transcription
repressors: decrease gene transcription
what are differences between promoters and enhancers
Promoters
- function within a short distance
- immediately upstream from the initiation site
- position-dependent: non-functional if moved
- orientation dependent: drive transcription in only one direction
Enhancers
- can function over a long distance
- can be upstream, downstream or within introns
- position independent: functional when moved
- orientation independent: function in normal or inverted orientation
How do enhancers communicate with promoters?
by bending DNA
